Disentangling the effects of selection and random genetic drift on natural genetic variation represents a central challenge in evolutionary biology. Many theoretical and empirical studies have focused on mutations that are 'slightly deleterious'with respect to fitness. The dynamics of these mutations are influenced by weak natural selection against them. However, particularly in small populations, the stochastic process of drift may allow these mildly harmful mutations to persist and eventually become fixed. Recent surveys of natural bacterial populations reveal that deleterious mutations are more prevalent than previously suspected;yet the interplay of forces governing the dynamics of these mutations remains largely unexplored. Since bacteria comprise a substantial fraction of Earth's biodiversity, this signals an enormous gap in our understanding of the processes that shape natural genetic variation. OBJECTIVES: The proposed project will clarify the evolutionary forces that influence the dynamics of deleterious mutations in bacteria, and will explore the consequences of these changes at the levels of populations, proteins, and whole genomes. We will test a key hypothesis from population genetic theory: a reduction in population size accelerates the accumulation of mildly harmful changes. We test this prediction in the context of different bacterial lifestyles. Specific Aims include (1) contrasting forces that shape protein evolution in host-dependent bacteria versus their free-living relatives, (2) examining how deleterious mutations shape host-microbe associations, including population-level variation and alterations in gene content, and (3) testing whether sequence-based inferences of deleterious substitutions correspond to reduced protein activities in lab-based assays. These investigations will distinguish the balance between selection and genetic drift in bacteria, with a particular focus on the influence of population size on the dynamics of deleterious changes. SIGNIFICANCE: Results of this project will elucidate modes of evolution among host-associated bacteria, including both pathogens and mutualists that live exclusively within eukaryotic host cells. The proposed studies of their molecular diversity and evolutionary dynamics will clarify the ability of these species to adapt to current hosts and to originate new host associations. By deciphering fundamental mechanisms that influence bacterial genome evolution, this study will contribute to a predictive framework for understanding how microbes evolve.